Fig 1: Hypothermic pre-conditioning of C2C12 myoblasts at 32 °C for 72 h followed by differentiation and respirometric analysis, with or without knockdown of RBM3.a Line graph showing the fold change of protein levels of RBM3 upon hypothermic treatment at 32 °C with respect to 37 °C at different time points (6 h and 48 h). b Western blot of protein levels of RBM3 using C2C12 cells cultured at 37 °C and 32 °C for 48 h and 72 h respectively. c Bar graph quantifying the western blot image in b, where the y-axis represents the intensity of RBM3 normalized to Actin (n = 4). Significance tested by two-tailed student’s t test (unpaired). d Western blot of protein levels of MHC using C2C12 cells transfected with scrambled (Scr) or siRBM3 and differentiated for 6 days at 37 °C after hypothermic pre-conditioning. e Bar graph quantifying the western blot image in d, where y-axis represents the intensity of MHC normalized to total protein, (n = 3). Significance tested by two-tailed student’s t-test (unpaired). f Line graph showing the mitochondrial oxygen consumption rate (OCR) of C2C12 cells transfected with Scr or siRBM3 and incubated at 37 °C and 32 °C (72 h) respectively. X-axis represents time in minutes and the y-axis represents oxygen consumption rate in pMol/min. Bar graph quantifying the basal respiration, maximum respiration, spare respiratory capacity and ATP-linked respiration of the cells (n = 4). Error bar in the bar graphs represent standard error of mean (SEM). Significance tested by two-tailed student’s t test (unpaired). *, **, *** p < 0.05, 0.01 and 0.001.
Fig 2: Analysis of metabolism, proliferation and differentiation of skeletal muscle myoblasts overexpressing RBM3 at 37 °C.a Line graph showing the mitochondrial OCR of C2C12 cells overexpressing RBM3-GFP and GFP (control) and respectively. X-axis represents time in minutes and the y-axis represents oxygen consumption rate in pMol/min. b Bar graph quantifying the basal respiration, maximum respiration, spare respiratory capacity and ATP-linked respiration of C2C12 cells overexpressing RBM3-GFP and GFP respectively. Y-axis represents oxygen consumption rate in pMol/Min (n = 3). Significance tested by two-tailed student’s t test (unpaired). c Heat map showing the intracellular levels of TCA metabolites in C2C12 cells overexpressing RBM3-GFP and GFP respectively (n = 3). d Box plot showing the intracellular levels of acetyl-CoA in C2C12 cells overexpressing RBM3-GFP and GFP respectively (n = 3). e EdU staining of C2C12 cells overexpressing RBM3-GFP and GFP respectively. Red color indicates EdU stained nucleus and blue color indicates DAPI staining. f Bar graph representing the quantitation of EdU staining where the y-axis represents the % of EdU positive nucleus (17 data points taken from 2 individual experiments). g Bar graph quantifying the % viability of C2C12 cells overexpressing RBM3-GFP and GFP respectively at 37 °C (n = 3). h Bar graph indicating cell proliferation of C2C12 cells overexpressing RBM3-GFP and GFP respectively at 37 °C where the y-axis represents the total number of cells, and the x-axis represents time in days (n = 3). Significance tested by two-tailed student’s t test (paired). i Western blot showing protein levels of MHC after 5, 6 and 7 days of differentiation using C2C12 myoblasts overexpressing RBM3-GFP and GFP respectively. j Bar graph quantifying the western blot in (i), where the y-axis represents the intensity of MHC normalized to the total protein (n = 3). k C2C12 myoblasts overexpressing RBM3-GFP and GFP respectively, differentiated for 6 days and immunostained with MHC antibody. l Bar graph quantifying the myotube fusion index (48 individual data points from 3 individual experiments) and myotube diameter of cells shown in (k) (28 individual data points from 2 individual experiments). m Primary myoblasts (from 3-month-old mice) overexpressing RBM3-GFP and GFP respectively, differentiated for 4 days and immunostained with MHC antibody. n Bar graph showing the myotube fusion index and myotube diameter quantified from the images shown in m (17 individual data points from 2 individual experiments). Error bar in the bar graphs represent standard error of mean (SEM). Significance tested by two-tailed student’s t test (unpaired). *, **, *** p < 0.05, 0.01, and 0.001 respectively.
Fig 3: Proteomic analysis of C2C12 myoblasts overexpressing RBM3 at 37°C.a Bar graph showing the number of upregulated, downregulated and unchanged proteins in C2C12 myoblasts overexpressing RBM3. b GO enrichment pathway analysis of upregulated proteins (n = 3). c GO enrichment analysis of protein-protein interaction networks of upregulated proteins (n = 3).
Fig 4: Proteomic analysis of C2C12 myoblasts overexpressing RBM3 at 37°C.a GO enrichment pathway analysis of downregulated proteins (n = 3). b GO enrichment analysis of protein-protein interaction networks of downregulated proteins (n = 3).
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